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The CHROMacademy Essential Guide Webcast:
What HPLC Operators Need to Know Part I
Everything Needed to Understand Mobile Phase, Pumping Systems and Autosamplers
 

Thursday 20th August 2015,
8:00am PDT / 11:00am EDT / 16:00 BST / 17:00 CEST

During this two part event we will look at the entire HPLC separation process from mobile phase selection and preparation, to injection, separation, and detection, from the perspective of the laboratory practitioner. We will provide practical hints and tips for the essential HPLC parameters in order to make your use of this powerful technique straightforward and successful.

Firstly we will discuss how and why mobile phases are chosen and prepared and the different variations of HPLC pumps and autosamplers which are available.

Column selection is probably one of the most important choices in any chromatographic method; therefore, we will share our insights on how to select the correct column each time covering particle size, morphology, physicochemical properties as well as stationary phase selection.

Finally, we will detail how to utilize and optimize HPLC gradients and review the various detectors available and when and how they are best employed.

Presented by Dr Paul Ferguson (Associate Principal Scientist, Astra Zeneca) and
Scott Fletcher (Technical BD Manager, Crawford Scientific).

Mobile Phase
  • How and why is a mobile phase pH chosen, controlled and measured?
  • Is filtration necessary?
  • Why are mobile phases degassed and which is the most efficient approach

Pumping Systems

  • What is the role and function of the (U)HPLC pump?
  • What are the differences and benefits of isocratic, binary and quaternary pumps?
  • What are the important major and minor operating variables

Autosamplers

  • How do modern HPLC and UHPLC autosamplers function
  • What are the critical parameters of an autosampler
  • How to optimize autosampler performance and spot problems

Column Selection (Part II)
Mobile Phase Gradients (Part II)

Detectors (Part II)


Key Learning Objectives:

  • Understand the HPLC process and which parameters can and should be considered
  • Learn how to select and prepare mobile phases appropriately
  • Appreciate the variations in HPLC pumps and their relative benefits
  • Gain knowledge on HPLC column particles and stationary phases
  • Understand how injections are made and how the autosampler can be optimized
  • Appreciate how and why gradients are used and how they can be effectively scaled and transferred
  • Understand how HPLC detectors operate and the specific analyte properties which lend themselves to the various detection techniques

Who Should Attend:

  • All analytical scientists
  • Anyone using HPLC or UHPLC
 
Biopharmaceutical Analysis
Sponsored by
 
 

Find out more about this Month's Essential Guide Webcast »

 
 

What HPLC Operators Need to Know Part I
Everything Needed to Understand Mobile Phase, Pumping Systems and Autosamplers

All operators of (U)HPLC instrumentation should possess significant knowledge and understanding in order to operate their instrumentation in the most efficient and effective manner. This mini-series of two webcasts will detail all critical parameters in setting up the instrument for operation, performing the analysis and ensuring the system has been left in a fully functioning capacity and ready for its next user.

Correct (U)HPLC set-up is essential, not only for the immediate analysis , but to ensure instrument down-time is reduced and up-time is increased. Good instrument operation always starts with appropriate mobile phase preparation. The majority of methods rely on the mobile phase being pH modified and controlled to facilitate acceptable peak shape and selectivity. Knowledge of how and why mobile phase pH’s are adjusted and effectively controlled is imperative to method robustness and system performance. How, when and why mobile phases are degassed and ensuring mobile phase compatibility with the prior system solvent is critical for baseline stability and peak response reproducibility. Correct mobile phase filtration is also pivotal to guard against pump wear and tear and avoid blockages.

The pump truly is the ‘heart’ of the instrument and its correct performance is vital for precise and accurate separations. Instrument operators should understand the components of the modern (U)HPLC pump and the role each element plays. The difference between isocratic, quaternary and binary pumps should be understood and specific modes by which each design function appreciated. Each design not only have their own individual benefits but also areas that may require additional set-up and optimization or maintenance. Finally, as the pump is hardest working, mechanically speaking, part of the instrument, it requires the greatest amount of maintenance. An effective preventative maintenance schedule is desired such that as much maintenance is planned prior to part component failure so that further components are alleviated and important chromatographic analysis are not compromised.

The injector introduces the sample into the mobile phase stream however the means by which this is accomplished is often overlooked by instrument operators. An understanding of all components and their individual and specific roles in this operation are beneficial as only when they’re fully appreciated can they be effectively optimized and maintained.

A good and competent (U)HPLC operator will have a sound understanding of mobile phase preparation and instrument set-up. They will also possess a good understanding of the pump and autosampler, not only they are optimized, but of any necessary maintenance, even if they are not directly involved in performing this.

Are you ‘fit for purpose’? This series of webcasts will ensure you are!

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eLearning Modules


Fast HPLC HPLC Pump

Not only is the pump operation imperative to achieve stable and reproducible peak responses but it is increasingly commonly employed in influencing selectivity by way of gradient analysis. The module will fully explain and demonstrate the key operating components of modern (U)HPLC pumps and also describe the different approaches to mobile phase gradient formation and compare and contrast their relative advantages and disadvantages.

Dual Piston Reciprocating Pump | Mixing Solvents – Binary Pumps | Mixing Solvents – Quaternary Pumps

 
Fast HPLC HPLC Autosamplers

An appreciation of the principle by which the modern (U)HPLC instrument introduces the sample into the mobile phase stream is often difficult to comprehend due to some of the critical operations being hidden from view. This module uses easy to follow and understand graphics and interactive animations to fully describe and explain the injection process.

Injection valve anatomy | Push to fill autosamplers | Integral fill autosamplers

 
 
Quick Guides

Fast HPLC HPLC Mobile Phases – 10 bad habits to avoid

Measuring the pH of the mobile phase after the organic has been added pH meters are calibrated to give the correct pH readback in aqueous solution – the buffers you verify this with are aqueous. If you measure the pH with the organic added, the pH will be different to that of measuring before organic addition. However, the most important point is to be consistent. If you do always measure pH after the organic is added, make sure you state this in the method so that everyone does it the same way. It won’t be 100% accurate, but at least it will be consistent. This is probably more important than having the exact pH.

HPLC Mobile Phases – 10 bad habits to avoid »

 
Fast HPLC HPLC Troubleshooting Guide to Cycling Baselines and Pressure Fluctuations

The Problem

A short term cycling baseline is seen along with pressure fluctuations...

Cycling Baselines and Pressure Fluctuations »

 
Fast HPLC HPLC Troubleshooting Guide to Peak Splitting Problems

The Problem

The analysis of an α2 adrenergic agonist and its main impurity are showing peak splitting that was not previously seen (Figure 1).  A moderate increase in system backpressure has also been observed.
The analyte is fairly hydrophobic with a log P value of 2.0.  The analyte possesses a number of basic nitrogen groups within its structure; however, the only significant pKa is at 7.49. 

Peak Splitting Problems »

 
Fast HPLC HPLC Filtration - Rocks, Boulders and Sand

First – what’s the point of filtration? Mainly to protect the HPLC system and analytical column from damage due to the build-up of particulate materials. Particulate build up usually results in an increase in system back pressure, to the point where the maximum system operating pressure is exceeded and the system shuts down...

HPLC Filtration »

 
Fast HPLC Setting HPLC Chromatographic Parameters

Whether you work in a regulated environment or not, setting chromatographic performance targets can help to keep us focussed.  Let’s consider how to set these targets and examine some real life examples that may not always follow the rules...

HPLC Chromatographic Parameters »

 
Fast HPLC Checklists are only for pilots?

How many times have you been in too much of a hurry to get to get a sequence running, set one tiny instrument parameter wrongly and had to spend the whole of the next day repeating the analysis...

HPLC Checklist »

 
 
Fast HPLC Critical Evaluation of HPLC Methods

There are numerous factors that affect the quality of your HPLC separation; including mobile phase composition, temperature, ionic strength, pH, column dimensions, and stationary phase and support material characteristics. These can all be manipulated to change and improve your HPLC separations. However, what are the changes we should expect? Will retention improve if another solvent is used and would we expect selectivity to change? Will certain analytes elute in the same order if the mobile phase pH, ionic strength, additives or temperature are changed?

Critical Evaluation of HPLC Methods »

 
Fast HPLC HPLC Troubleshooting - Eluents and Solvent Delivery Systems

This session considers important practical concepts such as good practice for eluent preparation, filtration and degassing as well as common related issues such as retention time drift and selectivity changes. We also explain the working principles of HPLC solvent delivery systems and highlight the components which are most susceptible to problems. The webcast concludes by considering various troubleshooting tests and strategies as well as common maintenance operations for solvent delivery systems.

Eluents and Solvent Delivery Systems »

 
Fast HPLC HPLC Troubleshooting - Autosampler, Column & Detector Issues

This session examines different autosampler designs, injection valve operations and common problems associated with modern HPLC autosamplers. Sample solvent and injection volume effects on chromatography will also be discussed in detail. We will consider column hardware design, materials of construction and associated issues – including minimizing dead volume and avoiding / troubleshooting column blockage issues, especially with the popular Sub 2μm column packing materials. We will also investigate the importance of controlling the temperature of the column during analysis and the potential chromatographic effects of poor temperature control. We will conclude by reviewing common HPLC detector hardware problems and the chromatographic issues associated with detector hardware and acquisition settings. We will build a checklist of suggested maintenance operations as well as outlining common diagnostic tests.

Autosampler, Column & Detector Issues »

 
 
 
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Key Learning Objectives:

  • Understand the HPLC process and which parameters can and should be considered
  • Learn how to select and prepare mobile phases appropriately
  • Appreciate the variations in HPLC pumps and their relative benefits
  • Gain knowledge on HPLC column particles and stationary phases
  • Understand how injections are made and how the autosampler can be optimized
  • Appreciate how and why gradients are used and how they can be effectively scaled and transferred
  • Understand how HPLC detectors operate and the specific analyte properties which lend themselves to the various detection techniques

Who Should Attend:

  • All analytical scientists
  • Anyone using HPLC or UHPLC

 

Dr. Paul Ferguson
Associate Principal Scientist
Astra Zeneca

 

Scott Fletcher
Technical BD Manager
Crawford Scientific